Human growth hormone (hereinafter referred to as “hGH”) is an aglycosylated peptide hormone that is secreted from the anterior pituitary gland and interacts with a specific receptor on the cell surface in various tissues so as to simulate the secretion of other growth factors, to account for increasing the size of various parts of the body. Ever since discovering that the growth hormone from the human pituitary gland is an effective therapeutic for pituitary dwarfism, hGH demand has increased explosively. However, the supply of hGH that can be extracted from the human pituitary gland is very limited. Further, after the incidence of the degenerative neurological disorder Creutzfeldt-Jacob disease in children who received cadaver-derived hGH, the FDA in the United States of America has banned the use of the hGH extracted from the pituitary glands of a cadaver, based on the assumption that infectious prions that caused the disease were transferred along with the cadaver-derived hGH (Roger, L., Science 234: 22, 1986). Currently, the biosynthetic human growth hormone produced by E. coli using gene recombination technology is commercially available with the approval of the FDA.
Polypeptides, such as hGH, are apt to degenerate due to their low stability, and are readily degraded by serum proteases and removed by the kidney or the liver. Hence, drugs containing polypeptides as pharmaceutically active ingredients have to be frequently administered to patients in order to maintain serum levels and titers thereof. However, the maintenance high serum levels of active polypeptides by frequent administration of protein drugs, which are in the form of injections in most cases, is painful to patients.
To solve these problems, attempts have been made towards maximizing medicinal effects by improving serum stability of protein drugs and maintaining a high serum level of protein drugs for a long period. Therefore, formulations of protein drugs that have increased stability and activity maintained at sufficiently high levels without inducing immune responses in patients, is needed.
For stabilizing proteins and preventing contact with protease and renal loss, conventionally, highly soluble polymers, such as polyethylene glycol (PEG), are chemically added to the surface of protein drugs. Being non-specifically conjugated to certain or various sites of target proteins, PEG can increase the solubility of the target proteins, stabilize the proteins and prevent them from being degraded, without causing significant side effects (Sada et al., J. Fermentation Bioengineering, 1991, 71:137-139). PEG conjugation can contribute to the stability of the proteins, but significantly decreases their activity. PEG of higher molecular weight has a lowered reactivity with proteins, thus reducing the yield.
An alternative strategy for increasing the in vivo stability of physically active proteins is by using genetic recombination to fuse the target proteins and the proteins that have high serum stability, which is a process of linking respective genes encoding the proteins to each other and culturing the animal cells transformed with the fused genes. For example, fusion proteins in which albumin or its fragments, known to increase the stability of proteins, fused to target proteins by genetic recombination have been reported (International Patent Publication Nos. WO 93/15199 and WO 93/15200, European Patent Publication No. EP 413,622).
U.S. Pat. No. 5,045,312 discloses that hGH conjugated with bovine serum albumin or murine immunoglobulin using a cross-linking agent has enhanced activity compared to the unmodified growth hormone. The only cross-linking agents mentioned in the patent are low-molecular weight compounds such as carbodiimide or glutaraldehyde. However, such low-molecular weight cross-linking agents do not guarantee homogeneous compositions due to their non-specific linkages, and may be toxic in vivo. Further, this patent revealed only the increase of activity of a growth hormone by chemical coupling, but did not exhibit the effect of chemical coupling on the activity on other polypeptide drugs, with no understanding of the correlation with the stability of proteins such as the increase in durability and serum half-life.
Recently, conjugates made between physiologically active polypeptides with an immunoglobulin Fc region and a non-peptide polymer have been introduced as long-acting formulations which promise protein drugs both in minimal reduction in activity and an increase in stability, as described in Korean Patent No. 10-0567902 (Physiologically Active Polypeptide Conjugate Having Improved In Vivo Durability), and Korean Patent No. 10-0725315 (Protein Complex Using An Immunoglobulin Fragment And Method For The Preparation Thereof).
According to these methods, hGH may be applied as the physiologically active polypeptide such that a long-acting hGH conjugate can be prepared. For these long-acting hGH conjugates to be used as drugs, it is essential that the in vivo medicinal effect of hGH be maintained while suppressing it from undergoing physicochemical changes such as denaturation, aggregation, adsorption or hydrolysis induced by light, heat or impurities in additives. Compared to hGH, a long-acting hGH conjugate is larger in size and molecular weight and thus is more difficult to stabilize.
Generally, proteins have a very short half life, and exhibit denaturation, such as the aggregation of monomers, precipitation by aggregation, and adsorption onto the surface of vessels, upon exposure to inappropriate temperatures, a water-air interface, high pressure, physical or mechanical stress, organic solvents, microbial contamination, etc. Once denatured, proteins lose their inherent physicochemical properties and physiological activity. Since protein denaturation is irreversible in most cases, it is almost impossible for denatured proteins to recover their inherent properties.
Absorbed proteins are apt to aggregate as they denature. The aggregated proteins may act as antigenic materials when injected into the body and, therefore proteins that are sufficiently stable must be administered. Various methods for preventing proteins from denaturing have been studied (John Geigert, J. Parenteral Sci. Tech., 43, No 5, 220-224, 1989, David Wong, Pharm. Tech. October, 34-48, 1997, Wei Wang., Int. J. Pharm., 185, 129-188, 1999, Willem Norde, Adv. Colloid Interface Sci., 25, 267-340, 1986, Michelle et al., Int. J. Pharm. 120, 179-188, 1995).
Some protein drugs adopted a lyophilization process to avoid the stability problems. However, lyophilized products are inconveniently dissolved in solvents for injection. Further, lyophilization requires a mass-scale freeze-drier, increasing the investment cost in the production of the protein drugs. Powdering of proteins with a spray drier was also suggested to maintain the stability of the proteins, but is not economically beneficial due to a low yield. Further, exposure to the high temperatures of spray drying produces negative side-effects on the proteins themselves.
Stabilizers, arising as an alternative approach overcoming these limitations, have been studied because when they are added to protein drug solutions, they have the ability to suppress physicochemical changes of protein drugs and guarantee in vivo medicinal efficacy even after long-term storage. Human serum albumin has been widely used as a stabilizer for various protein drugs, and the performance thereof has been proven (Edward Tarelli et al., Biologicals (1998) 26, 331-346).
When administered with human serum albumin, patients run the risk of being exposed to biological contaminants or pathogens such as mycoplasma, prions, bacteria and viruses because although the process used to purify albumin comprises the inactivation, screening or inspection of such biological contaminants or pathogens, these cannot be perfectly eliminated or inactivated. For example, a screening process comprises the inspection of donor's serum for certain viruses, but the inspection is not always reliable. Particularly, a very small number of certain viruses, if present, cannot be detected.
Due to their chemical differences, different proteins may be gradually inactivated at different rates under different conditions during storage. That is to say, the extension of the storage term by a stabilizer is not identical for different proteins. For this reason, stabilizers to be used vary in ratio to target proteins, concentration, and type depending on the physicochemical properties of the target proteins. Contrary to expectations, stabilizers, when used in combination, may bring about negative effects because of competition and interaction therebetween. Further, since the nature or concentration of target proteins may change during storage, the stabilizers used may provide effects different from those intended. Thus, a great amount effort and precautions are required to stabilize proteins in solutions.
Particularly, long-acting hGH conjugates that are prepared by linking the physiologically active peptide hGH with immunoglobulin Fc regions to improve the in vivo durability and stability of hGH require special compositions for stabilizing the protein because they are quite different in molecular weight and size from typical hGH.
International Patent Publication No. WO93/19776 discloses a stable liquid formulation of hGH comprising a buffer of pH 6.0˜7.0, an amino acid, mannitol and optionally a preservative such as benzyl alcohol. International Patent Publication No. WO94/03198 discloses a stable liquid formulation containing hGH, a buffer of pH 6.0, a non-ionic surfactant, a preservative, and, optionally, a neutral salt or mannitol. U.S. Pat. No. 6,448,225 discloses a stable pharmaceutically acceptable liquid formulation containing hGH, a buffer of pH 6.0, a non-ionic surfactant, and, optionally, a neutral salt or mannitol, with no glycine requirement. Korean Patent No. 10-0537260 discloses a stabilized liquid formulation of hGH containing PEG, instead of a non-ionic surfactant and a preservative, a buffer and an isotonic agent as active ingredients because non-ionic surfactants and preservatives cause hGH to be deaminated significantly.
However, hGH and an immunoglobulin Fc region, although both are peptides or proteins, have different physicochemical properties, and are both required to be stabilized at the same time. As illustrated above, different proteins may be gradually inactivated at different rates under different conditions during storage due to their chemical differences. Contrary to expectations, the use of stabilizers suitable for use in stabilizing peptides or proteins in combination may bring about negative effects because of the competition and interaction therebetween. Hence, as for long-acting hGH conjugates, the compositions of their stable formulations are different from those of formulations for stabilizing hGH alone. In fact, it is very difficult to discover a formulation for stabilizing both hGH and an immunoglobulin Fc region.
Leading to the present invention, intensive and thorough research into the safe storage of long-acting hGH-immunoglobulin Fc conjugates over a long period of time, conducted by the present inventors, resulted in the finding that a stabilizer composition comprising a buffer of pH 5.0˜6.0, a non-ionic surfactant, a sugar alcohol and a salt can provide an economically beneficial liquid formulation with a long-acting hGH conjugate which can give a great boost to increasing the stability of the long-acting hGH conjugate during storage for a long period of time without concerns about viral contamination.